This proposal is aimed at understanding the molecular functions of the multiple isoforms of tropomyosin in the reorganization of microfilaments following oncogenic transformation of cultured cells. We have demonstrated that tropomyosins with high Mr values are replaced with tropomyosins with low Mr values concomitant with the morphological alterations associated with a variety of cultured rat cell transformations. Furthermore, we have found two new actin-bundling proteins control the binding of the low Mr tropomyosins, and see how changes in tropomyosin-binding regulate the assembly of microfilaments upon cell transformation. Using limited proteolysis we plan to dissect the actin-bundling proteins, as well as those of the tropomyosins. We will microinject the two new actin-bundling proteins, high and low Mr tropomyosins, and the monoclonal antibodies, into living cells to see if the two actin- bundling proteins stimulate or inhibit the binding of low Mr tropomyosins to the actin bundles of stress fibers, and to determine if changes in tropomyosin-binding alter the organization of microfilaments and microtubules. Finally, we propose to undertake an in depth analysis of the structures and molecular compasitions of microfilaments and microtubules to determine if they change upon cell transformation. We will examine if such changes are functionally related to the changes in tropomyosin patterns. Our long-term goal is to understand how differential expression of tropomyosin effects changes in cell morphology, and why it is a very sensitive indicator of the transformed phenotype.